Most bacteria contain suicidal genes whose expression leads to growth arrest and eventual death upon exposure to cellular stress (reviewed by Elenberg-Kulka and Gerdes, Ann. Rev. Microbiol. 53: 43-70 (1999); Engelberg-Kulka et al., Trends Microbiol. 12: 66-71 (2004)). These toxin genes are usually co-expressed with their cognate antitoxin genes in the same operon (referred to as an addiction module or antitoxin-toxin system). E. coli has five addiction modules (Christensen et al., J. Mol. Biol. 332: 809-19 (2003)) among which the MazE/MazF module has been most extensively investigated. The x-ray structure of the MazE/MazF complex (Kamada et al., Mol. Cell. 11: 875-84 (2003)) is known and the enzymatic activity of MazF has been recently characterized (Zhang et al, J. Biol. Chem. 278: 32300-306 (2003)).
MazF is a sequence-specific endoribonuclease that specifically cleaves single-stranded RNAs (ssRNAs) at ACA sequences. An endonuclease is one of a large group of enzymes that cleave nucleic acids at positions within a nucleic acid chain. Endoribonucleases or ribonucleases are specific for RNA. MazF is referred to as an mRNA interferase since its primary target is messenger RNA (mRNA) in vivo. Transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs) appear to be protected from cleavage because of either their secondary structure or association with ribosomal proteins, respectively. Therefore, MazF expression causes nearly complete degradation of mRNA, leading to severe reduction of protein synthesis and ultimately, to cell death (Zhang et al., Mol. Cell. 12: 913-23 (2003)). MazF is found in selected bacteria, and recently the E. coli protein PemK (encoded by plasmid R100) was also shown to be a sequence-specific endoribonuclease (Zhang et al., J. Biol. Chem. 279: 20678-20684 (2004)). PemK cleaves RNA with high specificity at a specific nucleic acid sequence, i.e., UAX, wherein X is C, A or U. See PCT/US2004/018571, which is incorporated herein by reference. These sequence-specific endoribonucleases are conserved, underscoring their essential roles in physiology and evolution. We refer to this family of sequence-specific endoribonuclease toxins as “mRNA interferases” (Zhang et al., J. Biol. Chem. 279: 20678-20684 (2004)).
In the present study, we have exploited the unique cleavage properties of MazF to design a single-protein production (SPP) system in living E. coli cells. Upon expression of a gene engineered to express an ACA-less mRNA without altering its amino acid sequence, high levels of individual target protein synthesis were sustained for at least for 96 hours while background cellular protein synthesis was virtually absent. Therefore, the toxic effect of MazF is directed at mRNA with minimal side effects on cellular physiology. In fact, despite their state of growth arrest, these cells retain essential metabolic and biosynthetic activities for energy metabolism (ATP production), amino acid and nucleotide biosynthesis and transcription and translation. In addition to demonstrating the efficacy of the SPP system for human and yeast proteins, the technology was also effective for overexpression of an integral inner membrane protein whose natural levels of expression are relatively low. The SPP system yields unprecedented signal to noise ratios that both preclude any protein purification steps for experiments that require recovery of proteins in isolation, and, more importantly, enable structural and functional studies of proteins in intact, living cells.
This bacterial single protein production (SPP) system supports high yield recombinant protein production in the virtual absence of background cellular protein synthesis. This high signal to noise ratio is facilitated by coexpression of an endoribonuclease that specifically cleaves ACA sequences of mRNAs (resulting in global mRNA degradation and translation inhibition) along with an ACA-less target gene (whose mRNA is uncleavable so its translation is undeterred). Now we have optimized the expression vectors and growth conditions to tailor this bacterial bioreactor technology toward highly economical protein production for structure determination by NMR and X-ray crystallography. We also demonstrate that exponentially growing cultures could be condensed 40-fold (cSPP) without affecting final protein yields and support very high incorporation of selenomethionine and fluorophenylalanine without cytotoxicity. cSPP also resulted in a substantial reduction in the cost of sample labeling, to only 2.5% that of conventionally prepared samples. This major cost efficiency, coupled with the absence of cytotoxicity upon robust protein expression, imparts advantages to the cSPP system that are especially well suited to the structural genomics mission and other large scale protein expression applications.